The functional roles of the circRNA/Wnt axis in cancer

CircRNAs, covalently closed noncoding RNAs, are widely expressed in a wide range of species ranging from viruses to plants to mammals. CircRNAs were enriched in the Wnt pathway. Aberrant Wnt pathway activation is involved in the development of various types of cancers. Accumulating evidence indicates that the circRNA/Wnt axis modulates the expression of cancer-associated genes and then regulates cancer progression. Wnt pathway-related circRNA expression is obviously associated with many clinical characteristics. CircRNAs could regulate cell biological functions by interacting with the Wnt pathway. Moreover, Wnt pathway-related circRNAs are promising potential biomarkers for cancer diagnosis, prognosis evaluation, and treatment. In our review, we summarized the recent research progress on the role and clinical application of Wnt pathway-related circRNAs in tumorigenesis and progression.


Background
Cancer is one of the main causes of death today and has become a serious public health problem worldwide [1][2][3][4][5]. It is a complex disease that involves changes in a variety of processes, including genetic and epigenetic characteristic changes [6][7][8]. The molecular changes in cancer genes and related signaling pathways could provide information for cancer diagnosis and targeted therapy [9][10][11]. This information could contribute to improvements in cancer diagnosis and treatment.

The wnt pathway in tumorigenesis
The Wnt gene was first identified in mouse mammary tumors in 1982 [74][75][76]. At that time, it was designated as int1 [75,77]. Because of the high homology between the mouse int1 gene and the Drosophila Wingless gene, the researchers merged Wingless with Int1 and assigned the name Wnt gene [78,79]. The Wnt gene, localized at 12q13, mediates physiological effects in a paracrine and autocrine manner [78,80]. The signaling pathways regulated by the Wnt gene are collectively termed the Wnt pathway. The Wnt signaling pathway is highly conserved from Drosophila to humans. The pathway [81][82][83] is critical for a wide variety of cellular functions, such as cell polarity, movement, proliferation, asymmetric division, and muscle tissue development. Wnts are a family of secreted, lipid-modified proteins that bind to Frizzled receptors to activate signaling cascades [84,85]. The Wnt pathway can be divided into three classes: Wnt/β-catenin signaling, Wnt/planar cell polarity signaling, and Wnt/ Ca signaling [86][87][88][89] (Fig. 1). Wnt/β-catenin signaling, a canonical Wnt signaling pathway, is involved in the regulation of gene expression [90][91][92]. Wnt/planar cell polarity signaling regulates cell polarity and directional cell movements [83,93,94]. Wnt/Ca signaling is obviously Fig. 1 Wnts are a family of secreted, lipid-modified proteins that bind to Frizzled receptors to activate signaling cascades. The Wnt pathway can be divided into three classes: Wnt/β-catenin signaling, Wnt/PCP signaling, and Wnt/Ca signaling associated with the release of intracellular calcium [95,96]. Dysregulation of the Wnt pathway has a strong relevance to cancer.

Wnt/β-catenin signaling
The Wnt/β-catenin signaling pathway is characterized by the cellular redistribution and nuclear accumulation of the β-catenin gene [97,98]. Wnt protein combines with Frz and LRP5/6 on the cell surface to form a trimer, which transmits the signal and activates the protein Disheveled [Dsh/DVL] [99,100]. This leads to the disassociation of the β-catenin degradation complex adenomatous polyposis coli (APC)/Axin/GSK-3β (glycogen synthase kinase 3β) and increases the cytoplasmic levels of β-catenin [101,102]. Then, upregulated β-catenin is transferred into the nucleus. Nuclear β-catenin interacts with T cell transcription factor (TCF])/lymphoid enhancer factor (LEF) and finally activates the expression of downstream target genes [98,[103][104][105]. The Wnt/β-catenin signaling pathway participates in the cancer process by acting as an important modulator [106][107][108] of cell proliferation, metastasis, and differentiation. Overexpression of the Wnt gene or mutation in one of the components that causes β-catenin degradation leads to activation of the Wnt/β-catenin pathway.

Wnt/PCP signaling
In the Wnt/PCP signaling pathway, Wnt binds to frizzled transmembrane receptors and then activates the protein Disheveled (Dsh/DVL), leading to a series of cell signaling cascades [109][110][111][112]. DSH is connected to the downstream effectors Rho and ROCK (Rho-associated kinase) through Daam1. RAC is directly activated by Dsh, and Dsh further activates JNK by activating mitogen-activated protein kinases (MAP3Ks) and MAP2Ks [113,114]. The PCP pathway is associated with cell polarity, cell alignment and cell migration.

Wnt/Ca 2 + signaling
In the Wnt/Ca 2 + pathway, the Wnt protein is mainly composed of Wnt1, Wnt5A and Wnt11 and binds to the Frizzled transmembrane receptor on the cell surface [115,116]. The combination of the Wnt protein and Frizzled activates Disheveled, which activates PLC through the G protein [117,118]. These cellular processes could finally promote the release of intracellular Ca2+. The activation of Disheveled could also activate the cGMPspecific phosphodiesterase PDE6 and reduce intracellular cGMP, which leads to an increase in the intracellular Ca2 + concentration [119][120][121][122][123]. Elevated cytoplasmic Ca2 + concentrations can stimulate the nuclear factor NFAT and other transcription factors [124,125]. These processes trigger the activation of downstream pathways and a series of altered cell functions. The Wnt/ Ca2 + pathway is essential for early embryonic development, interneural communication and the inflammatory response [126,127].

CircRNA in the wnt pathway
CircRNAs, first found in the 1890s [128], remain enigmatic owing to technological limitations and limited existing knowledge. In 2013, Hansen TB et al. first proposed and confirmed that circRNAs function as miRNA sponges [129]. This finding started a new era in circRNA research [32,42,[130][131][132][133]. Unlike linear RNAs, circR-NAs are single-stranded, covalently closed noncoding RNAs without 5' end caps or 3' end poly (A) tails [25][26][27][28]. CircRNAs are not affected by RNA exonuclease, and their expression is more stable [134,135]. CircRNAs are formed by reverse splicing events [29][30][31]. A mechanistic model argued that the RNA is partially folded during the transcription of pre-RNA. Initially, nonadjacent exons are pulled closer by RNA folding, and exon skipping occurs. The spanned region forms a circular RNA intermediate, and then circRNAs are formed by further splicing. Another model suggests that the reverse complement sequence located in the intron region causes the intron region to pair and mediate reverse splicing to form circRNA [136][137][138][139][140].
CircRNAs act mainly through four molecular mechanisms. In regulating gene expression, circRNAs affect the expression of parental gene mRNA by interacting with RNA binding proteins [141][142][143]. Competitive complementary pairing between introns can strike a balance with linear RNAs during the formation of circR-NAs. CircRNAs can also exert their functions by acting as competing endogenous RNAs (ceRNAs) of miRNAs [144][145][146][147]. In addition, circRNAs are involved in the immune response [29,148,149]. Endogenous circRNAs play a role in the antiviral response, while exogenous circRNAs can stimulate immune signaling in mammalian cells by activating the pattern recognition receptor RIG-L [150][151][152][153]. Moreover, although circRNAs are noncoding RNAs, a few circRNAs can also perform regulatory functions by encoding peptides [154][155][156]. Several previous studies have shown that circRNAs play an important role in tumorigenesis and tumor progression. CircRNA_403658 facilitates aerobic glycolysis and cell growth by upregulating LDHA expression in bladder cancer [157]. CircRNA_103809 functions as an oncogene in the progression of hepatocellular carcinoma [158].
Both circRNAs and the Wnt pathway play a critical role in cancer development and progression. CircRNAs negatively or positively regulate cancer initiation, promotion, and progression by directly or indirectly interacting with the Wnt pathway. The interaction of circRNAs and the Wnt pathway has a noticeable impact on cell growth, metastasis, and other malignant cell behaviors in cancer. The majority of circRNAs act as sponges of miRNAs to activate or inactivate the Wnt pathway. With the deepening of research, more action modes between circRNAs and the Wnt pathway will be found. Related studies are expected to provide new insights for the diagnosis and treatment of cancer.

The role of the circRNA/Wnt axis in cancer
CircRNAs related to the Wnt pathway are aberrantly expressed in many cancers. Emerging evidence suggests that a range of clinical characteristics have been associated with circRNAs related to the Wnt pathway (Table 1). Moreover, the circRNA/Wnt axis contributes to cancer progression by modulating many cell biological functions. In this section, we will introduce the expression, corresponding clinical features, functions and mechanisms of the circRNAs/Wnt axis ( Table 2).

Digestive tumors Esophageal cancer
Elevated levels of circRNA_100367 were observed in radioresistant esophageal cancer cell lines [192], while the expression of cir-ITCH was downregulated in esophageal squamous cell carcinoma (ESCC) tissues [193] (Fig. 2). The expression of cir-ITCH is positively associated with linear ITCH in ESCC. Functionally, colony formation and Cell Counting Kit-8 (CCK-8) assays showed that cir-ITCH could inhibit ESCC tumor growth through the regulation of cell proliferation. Knockdown of cir-cRNA_100367 attenuates cell proliferation, migration, and radioresistance in esophageal cancer [192]. cir-cRNA_100367 decreases radiation sensitivity by regulating the miR-217/Wnt3 pathway. CircRNA_100367 could also affect esophageal cancer cell growth under irradiation in vivo. Using bioinformatics tools, Su et al. [234] found that a large number of circRNAs were closely related to cancer progression. Further studies on these molecules are still required.
Interestingly, some researchers found that the expression of circCNIH4, cir-ITCH, and circ_0001649 was significantly downregulated in gastric cancer tissues and cells [160,196,198]. cir-ITCH is closely related to lymph node metastasis and patient prognosis [160]. CircCNIH4, cir-ITCH, and circ_0001649 markedly reduced cell proliferation, migration, and invasion in gastric cancer cell lines. CircCNIH4 and circ_0001649 also contribute to gastric cancer progression through the regulation of cell apoptosis [196,198]. CircCNIH4 inhibits the Wnt/βcatenin pathway by upregulating DKK2 and FRZB levels ( Fig. 3). Similarly, cir-ITCH reduce miR-17 levels to inactivate the Wnt/β-catenin pathway. Circ_0001649 inhibits the ERK and Wnt/β-catenin signaling pathways by sponging miR-20a.
On the other hand, the expression of circ_0004018, circ_0003418, and circ-ITCH is significantly downregulated in HCC [169,170,174,208]. CircZKSCAN1 and circ-ITCH are potential prognostic biomarkers [171,174]. Circ_0004018 and circ_0003418 are negatively correlated with tumor size [169,170]. In addition, the expression of circ_0003418 has been reported to be related to TNM stage and HBsAg levels in HCC [170]. Circ_0004018 and circ_0003418 contribute to cancer development and progression by regulating many cell biological functions, including cell proliferation, migration, and invasion. Knockdown of circZKSCAN1 could inhibit the malignant behaviors of HCC cancer stem cells, such as sphere formation, colony formation, cell proliferation, and metastasis. Circ_0004018 modulates the Wnt/ β-catenin pathway to accelerate HCC progression by targeting the miR-626/DKK3 axis. CircZKSCAN1 binds with FMRP to increase Wnt signaling activity in HCC.

Nervous system neoplasms Glioma
Malignant gliomas are the most common primary tumors of the central nervous system [256][257][258][259]. Wnt pathway-associated circRNAs have drawn much attention in glioma research in recent years [260][261][262][263]. The levels of circ_0001730, circKIF4A, circ_0000177, and cZNF292 are upregulated in glioma [182,183,217,218,264] tissues versus normal brain tissues. Circ_0000177 is related to clinical stage, and patients with increased circ_0000177 expression have a poor prognosis [183]. Circ_0001730, circKIF4A, and circ_0000177 are all involved in tumor cell growth and metastasis in glioma [182,183,217]. cZNF292 promotes cancer development by regulating cell proliferation, the cell cycle, and angiogenesis. Mechanistically, circ_0001730 functions as a sponge of miR-326 to positively regulate Wnt/βcatenin pathways in the pathophysiologic processes of glioma. Circ_0001730 could also be upregulated by SP1 [218]. Overexpression of circ_0000177 increases FZD7 levels to activate Wnt signaling mediated by miR-638 in glioma.

Genitourinary tumors Prostate cancer (PCa)
PCa refers to an epithelial malignancy that occurs in the prostate [265][266][267][268][269]. The expression of cir-ITCH was significantly downregulated in PCa tissues and cell lines [219]. Further experiments showed that cir-ITCH could attenuate PCa cell viability and invasion. Cir-ITCH hinders PCa development by inactivating the Wnt/β-Catenin and PI3K/AKT/mTOR pathways. Not much is known about Wnt pathway-associated circRNAs in PCa. There is a crucial need for Wnt pathway-associated circRNA research in PCa [219].

Female reproductive system cancers
Cancers that originate in the female reproductive system are called female reproductive cancers [270]. Ovarian cancer (OC), endometrial cancer (EC), and cervical cancer are the three most common gynecological malignancies [271][272][273][274]. The expression of circ-ABCB10 is significantly upregulated, while circPLEKHM3 expression is downregulated in OC [184,220]. Moreover, the level of circPLEKHM3 is positively associated with the overall survival rate in patients with OC [184]. Circ-ABCB10 remarkably facilitates cell proliferation and invasion and reduces cell apoptosis by miR-1271 in OC [220]. Circ-ABCB10 plays a critical role in OC progression via the regulation of Capn4/Wnt/β-catenin. CircPLE-KHM3 inhibits cell proliferation and migration by sponging miR-9 and regulating the BRCA1/DNAJB6/KLF4/ AKT1/Wnt/β-catenin axis in OC [184]. Circ_0109046 and circ_0002577 are elevated in EC tissues and cell lines [185,186]. The overexpression of circ_0002577 is positively correlated with advanced FIGO stage and lymph node metastasis in EC. High expression of circ_0109046 and circ_0002577 predicts a poor prognosis in patients with EC. Circ_0109046 activates the Wnt/β-catenin pathway by sponging miR-105 to increase SOX9 levels. Circ_0002577 functions as a sponge of miR-197 to regulate the CTNND1/Wnt/β-catenin axis in EC. Circ-SAMD11 expression is markedly upregulated in cervical cancer [221]. Silencing of circSAMD11 expression suppressed cell proliferation and metastasis and promoted cell apoptosis in cervical cancer. The circSAMD11/miR-503/sox4/Wnt/β-catenin axis plays an essential role in the progression of cervical cancer [221].

Tumors of the blood system
Hematological malignancies, also known as neoplasms of the blood, lymph nodes and bone marrow, include leukemia, lymphoma, and multiple myeloma [275][276][277][278]. The common types of leukemia are acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic Fig. 4 Wnt pathway-associated circRNAs in respiratory system cancer, nervous system cancer, genitourinary tumors, blood system cancers, musculoskeletal system cancer, endocrine system cancer, and cancers of other systems lymphocytic leukemia (CLL) [279][280][281][282]. Circ_0121582 expression is significantly decreased in AML [222]. Functional experiments demonstrated that the overexpression of circ_0121582 significantly attenuated cell survival and promoted the cell cycle arrest in AML. Circ_0121582 activates Wnt/β-catenin by sponging miR-224 to increase GSK3β expression in AML. The expression of circ-CBFB was upregulated in CLL, and it has been reported as an independent predictive factor for the prognosis of CLL [223]. Circ-CBFB facilitates CLL cell proliferation and inhibits cell apoptosis by sponging miR-607 and upregulating the FZD3/Wnt/βcatenin axis. Diffuse large B-cell lymphoma (DLBCL) is the most common malignant lymphoma subtype [283][284][285]. The level of circ-APC is significantly decreased in the tissues, cell lines, and plasma of DLBCL patients versus normal controls [224]. circ-APC inactivates the Wnt/β-catenin pathway to suppress cell proliferation in DLBCL through the regulation of the miR-888/APC and TET1/APC axes.

Tumors of the musculoskeletal systems Osteosarcoma (OS)
OS is the most common primary malignant neoplasm of the bone and mainly affects children, adolescents, and young adults [286][287][288][289]. The level of circMYO10 is significantly elevated [63], while circ_0002052 expression is downregulated in OS tissues and cell lines [187]. The expression of circ_0002052 is positively associated with overall and progression-free survival in patients with OS. circ_0002052 inhibits cell growth and cell motility and enhances cell apoptosis in OS by sponging miR-1205 and modulating the APC2/Wnt/β-catenin axis. CircMYO10 functions as an oncogene in OS progression. The overexpression of circMYO10 facilitates OS cell proliferation and EMT in vitro. CircMYO10 facilitates histone H4K16 acetylation by regulating the miR-370-3p/RUVBL1 axis and activating Wnt/β-catenin signaling in OS. Cisplatin (DDP) is a conventional chemotherapy drug in the treatment of OS [290][291][292][293]. Cisplatin resistance is a major challenge for OS chemotherapy application [294,295]. CircUBAP2 expression is increased in cisplatin-resistant OS tissues and cells [225]. Silencing circUBAP2 inhibits cell proliferation, migration, and invasion and induced apoptosis in OS. CircUBAP2 knockdown also suppresses cisplatin resistance by regulating miR-506-3p/SEMA6D and the Wnt/β-catenin pathway [225].

Tumors of the endocrine system Thyroid cancer
The incidence rate of thyroid cancer has been increasing throughout the world [296][297][298][299][300]. CircRNA_102171 and circRNA_NEK6 are relatively upregulated [226,227], while circ-ITCH is downregulated in thyroid cancer tissues and cell lines [188]. The level of circ-ITCH is closely associated with clinical stage, lymph node metastasis, and patient prognosis in thyroid cancer. CircRNA_102171 and circRNA_NEK6 play a promoting role in cell growth and metastasis. CircRNA_102171 activates the Wnt/βcatenin pathway in a CTNNBIP1-dependent way [226]. CircRNA_NEK6 facilitates thyroid cancer progression by sponging miR-370-3p and upregulating the FZD8/Wnt axis [227]. Circ-ITCH exerts its tumor suppressor action by modulating miR-22-3p/CBL/β-catenin in thyroid cancer [188].

CircRNA, a potential biomarker in wnt pathway
Despite technological advances, cancer diagnosis and treatment are still a challenge that may require the emergence of new tumor biomarkers [310,311]. Increasing evidence has revealed that Wnt-associated circRNAs are closely related to cancer progression. Wnt-associated circRNAs may be very promising biomarkers in cancer diagnosis, prognosis, and treatment. In this section, we will further discuss their potential application in clinical practice.

Diagnosis
The early screening and diagnosis of cancer is conducive to the survival of cancer patients [312][313][314][315][316]. Identifying suitable biomarkers has always been a difficult issue in cancer research. Wnt-associated circRNAs may be used to assist early diagnosis in many cancers. They are aberrantly expressed in many kinds of tumors from multiple systems, such as digestive tumors, respiratory system tumors, nervous system neoplasms, genitourinary tumors, musculoskeletal system tumors and endocrine system cancers. Moreover, plasma circ-APC levels are significantly downregulated in DLBCL [224]. This discovery indicates a more convenient clinical application of circ-APC as a diagnostic marker. Studies further evaluated the diagnostic potential for cancer by receiver operating characteristic (ROC) curve analysis. Yang et al. found that the AUC value of circ0005654 was 0.781 in gastric cancer [159]. ROC analysis of circRASSF2 expression levels in colorectal cancer tissues and cells accurately discriminated between CRC patients and healthy controls (AUC: 0.9863) [162]. Further experimental verification and research on circRASSF2 in body fluids is necessary. The corresponding AUC value for circ-CBFB was 0.80 in chronic lymphocytic leukemia [223].

Prognosis prediction
Early prognostic information is important in making treatment decisions [317][318][319][320][321]. A growing amount of evidence shows that Wnt-associated circRNAs can be of important prognostic value. These circRNAs are closely related to overall survival, disease-free survival, recurrence-free survival, 5-year survival rate, and progression-free survival in several cancers. Patients with lower circZKSCAN1 expression have shorter overall and recurrence-free survival in HCC [171]. Li et al. [166] reported that the overexpression of circCCT3 was negatively correlated with the disease-free survival rate in colorectal cancer. Higher circ_0109046 expression predicts a decreased 5-year survival rate in patients with endometrial carcinoma [185]. Such studies have important implications in prognosis evaluation and treatment selection. In addition, Wnt-associated circRNAs are associated with other relevant prognostic factors. For example, downregulated circMTO1 levels predict advanced TNM stage and lymph node metastasis in CRC [70].

Cancer treatment
Despite rapidly progressing treatment modalities, cancer therapy remains one of the most challenging issues in the world. CircRNA-based targeted therapeutic strategies shed new light on the evolution of cancer treatment [42,43,262,322,323]. CircRNAs regulate many cell biological functions by directly or indirectly interacting with the Wnt pathway. CircRNA_NEK6 activated the FZD8/Wnt axis to facilitate thyroid cancer progression by sponging miR-370-3p [227]. Circ_0121582 promotes GSK3β expression to activate the Wnt/β-catenin pathway by sponging miR-224 in AML [222]. Circ-SFMBT2 contributes to the development and tumorigenesis of gastric cancer via regulation of the miR-1276/CTNNB1/Wnt/βcatenin axis [195]. Controlling Wnt-associated circRNA expression may be an effective approach for cancer treatment. The knockdown of circ_SMAD4 blocked gastric cancer progression by negatively regulating cell growth [73]. Silencing circ-ZNF124 expression inhibited malignant phenotypes in NSCLC cells [73]. In addition, Circ-ITCH is a tumor suppressor in many cancers [160,174,181,188,190,193,207,208,219]. Wang et al. found that upregulated circ-ITCH expression suppressed cell proliferation and invasion in papillary thyroid cancer [188]. However, the identification of targeted drugs that can stably control the expression of circRNA and transmit this effect is the current difficulty. This requires a deeper understanding of the structure and function of Wnt-associated circRNAs. The majority of circRNAs act as sponges of miRNAs to activate or inactivate the Wnt pathway. Regulating the target miRNAs of Wntassociated circRNAs may also be feasible. MiR-582 intervention effectively reversed the cell biological functions regulated by circ_0009361 in CRC [205].

Conclusions and future perspectives
The Wnt signaling pathway is highly involved in cancer development, and essential for a wide variety of cellular functions, such as cell polarity, movement, proliferation, asymmetric division, and muscle tissue development. Both circRNA and the Wnt pathway play a critical role in cancer development and progression. Emerging data suggest that the circRNA/Wnt axis modulates the expression of cancer-associated genes and then regulates tumor progression. CircRNAs are enriched in the Wnt pathway. Wnt-associated circRNAs are abnormally expressed in digestive tumors, respiratory system tumors, nervous system neoplasms, genitourinary tumors, musculoskeletal system tumors, endocrine system cancers and other cancers. Their aberrant expression indicates their potential as diagnostic markers. However, most related experiments are based on tissue and cell research. Ideal and effective molecular markers should be stably expressed in plasma, serum, and other body fluids. Such molecules have greater potential for clinical applications. Wnt-associated circRNAs are also promising potential biomarkers in the treatment of cancer. CircRNAs negatively or positively regulate cancer initiation, promotion, and progression by directly or indirectly interacting with the Wnt pathway. We could enhance the expression of cancerpromoting circRNAs or inhibit the expression of tumor suppressor circRNAs to control cancer progression. The current goal is to find targeted drugs that can stably control the expression of circRNA and induce this effect. We need to further understand the structure and function of Wnt-related circRNAs. Furthermore, the interaction and the related mechanisms between circRNAs involved in the Wnt pathway need more studies to confirm.